This is a proposal to develop a high throughput assay system to detect inhibitors of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) formation and/or maintenance. HBV cccDNA is essential to the virus life cycle and its elimination during chronic infection is considered critical to durable therapy. However, because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself, cccDNA has not previously been targeted in high throughput screening of small molecule libraries. In this proposal, a tissue culture line that expresses reporter molecules that are produced in a cccDNA-dependent manner is created. This cell line will produce viral transcripts from a stably integrated, linear, inducible HBV genome (transgene), leading to transcription of full-length viral pregenomes and cccDNA formation. The critical feature will be that the N and C termini of the open reading frame (ORF) for the viral "e antigen" (HBeAg) are at opposite ends of the integrated HBV transgene. The formation of cccDNA from pregenomic transcripts will unite the divided ORF. Therefore, intact HBeAg RNA and protein will only be made from transcripts produced from the circular, cccDNA templates. This is feasible, since in current, early, versions of this cell line, detection of HBeAg correlates with cccDNA levels. In an HTS campaign, compounds that lower HBeAg, as detected by an immunological assay, would be considered candidates for cccDNA inhibition. Other reporter molecules are also proposed, and secondary assays to validate [unreadable]hits[unreadable] as true inhibitors of cccDNA will be designed and implemented. Prototype cell lines, meeting most of the needed criteria, are already in hand. Thus, our assay, and the inhibitors identified by it, would be novel and have great therapeutic and research potential.